Stem Cell

Sphingolipids are important structural components of cellular membranes. They also function as prominent signaling molecules to control a variety of cellular events, such as cell growth, differentiation, and apoptosis. Impaired sphingolipid metabolism, particularly defects in sphingolipid degradation, has been associated with many human diseases. Fluorescence sphingolipid analogs have been widely used as efficient probes to study sphingolipid metabolism and intracellular trafficking in living mammalian cells. Compared with nitrobenzoxadiazole fluorophores (NBD FL), the boron dipyrromethene difluoride fluorophores (BODIPY FL) have much higher absorptivity and fluorescence quantum. These features allow more intensive labeling of cells for fluorescence microscopy imaging and flow cytometry analysis. Here, we describe a protocol employing BODIPY FL-labeled sphingolipid analogs to elucidate sphingolipid internalization, trafficking, and endocytosis in mouse embryonic stem cells.

Graphical abstract:

In many fields of biology, especially in the field of developmental biology, LacZ reporter staining is an approach used to monitor gene expression patterns. In the LacZ reporter system, the LacZ gene is inserted in the endogenous location of the target gene via gene knock-in or by constructing a transgenic cassette in which LacZ is placed downstream of the promoter of the target gene being examined. Currently, the most common LacZ staining methods used are X-gal/FeCN staining and S-gal/TNBT staining. A serious limitation of both of these methods is that they are not effective when the LacZ gene is expressed at a low level. In an attempt to remedy this problem, we have established a new staining protocol which combines both methods. When compared to them, the method described here is better for visualizing lowly expressed genes and it has low background with high sensitivity.

Two main features characterize pluripotent cells; self-renewal (unlimited cell division) and the ability to give rise to all cells of the adult organism. Given the recent impact of induced pluripotent stem cells (iPSCs) and ongoing use of pluripotent embryonic stem cells ESCs (ESCs) in basic discovery, drug development, and potential use for stem cell therapy and regenerative medicine, methods to definitively distinguish pluripotent cells from their differentiated derivatives are required. This will allow us to better understand the factors that promote their survival, self-renewal, and lineage-specific differentiation.

Undifferentiated embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) may be identified through the use of biomarker and functional assays. Biomarker assays include those for transcript and protein expression of important pluripotency transcription factors (OCT4, SOX2, and NANOG), cell surface markers (SSEA-1, -3, and -4; TRA-1-60, TRA-1-81), and Alkaline Phosphatase (AP) activity (Brambrink et.al., 2008; Ginis et al., 2004). Functional assays include: (1) the ability to generate teratomas consisting of cells from all three germ layers (endoderm, ectoderm, and mesoderm) when transplanted into immunodeficient mice or upon in vitro differentiation; (2) the ability to generate a chimera; and (3) germline transmission (Marti et al., 2013; Buehr et al., 2008). The latter two tests are ethically feasible only for mouse and other non-human pluripotent cells.

In this protocol (Campbell and Rudnicki, 2013) we describe a rapid method to screen for pluripotent cells by AP activity. AP, also known as Basic Phosphatase catalyzes the dephosphorylation of many molecules including nucleotides and proteins. AP activity is high in pluripotent cells but is greatly decreased in more differentiated cell types. The technique described herein may be used to enumerate pluripotent cells during differentiation in the presence or absence of specific genetic manipulations or small chemical modulators. It may also be used to monitor induced pluripotency using defined factors from more differentiated cell types.